Epothilons C and D, preparation and compositions

ABSTRACT

The present invention relates to epothilon derivatives and to their use.

CROSS REFERENCE TO RELATED APPLICATION

This a divisional of Ser. No. 09/836,134, filed Apr. 16, 2001, now U.S. Pat. No. 6,613,912 B2 issued Sep. 2, 2003, which in turn is a divisional of Ser. No. 09/077,055, filed Aug. 3, 1998, now U.S. Pat. No. 6,288,237 B1 issued Sep. 11, 2001, the latter corresponding under 35 U.S.C. §371 to PCT/EP96/05080, filed Nov. 18, 1996.

The present invention relates generally to epothilon derivatives and to their use in the preparation of medicaments. The present invention relates especially to the preparation of epothilon derivatives of general formulae 1 to 7 shown hereinafter and to their use in the preparation of therapeutic compositions and compositions for plant protection.

In formulae 1 to 7 given above:

R═H, C₁-₄alkyl;

R¹, R², R³, R⁴, R⁵═H, C₁₋₆alkyl,

C₁₋₆acyl-benzoyl,

C₁₋₄trialkylsilyl,

benzyl,

phenyl,

benzyl or phenyl each substituted by C₁₋₆alkoxy,

C₆alkyl, hydroxy or by halogen;

it also being possible for two of the radicals R¹ to R⁵ to occur together to form a group —(CH₂)_(n)— wherein n is from 1 to 6, and the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals;

Y and Z are either identical or different and each represents hydrogen, halogen, such as F, Cl, Br or I, pseudohalogen, such as —NCO, —NCS or —N₃, OH, O—(C₁₋₅)acyl, O—(C₁₋₆)alkyl, O-benzoyl. Y and Z may also be the O atom of an epoxy group, epothilon A and B not being claimed, or one of the C—C bonds forms a C═C double bond.

In formula 3, X generally represents —C(O)—, —C(S)—, —S(O)—, —CR¹R²—, wherein R¹ and R² are as defined above, or —SiR₂— wherein R is as defined above.

In formula 4, X represents oxygen, NOR³, N—NR⁴R⁵ or N—NHCONR⁴R⁵, wherein the radicals R³ to R⁵ are as defined above.

In formula 5, X represents hydrogen, C₁₋₁₈alkyl, C₁₋₁₈acyl, benzyl, benzoyl or cinnamoyl.

For epothilons A and B, see DE-A-41 38 042.

Compounds according to general formula 1 can be obtained starting from epothilon A and B and from their 3-O— and/or 7-O-protected derivatives by opening the 12,13-epoxy group. If hydrohalic acids are used for that purpose in a preferably non-aqueous solvent, there being obtained the halohydrins X═Hal, Y═OH and Y═OH, Y═Hal. Protonic acids, such as, for xample, toluenesulphonic acid and trifluoroacetic acid, result, in the presence of water, in 12,13-diols which are then acylated (e.g. with carboxylic acid anhydrides and pyridine or triethylamine/DMAP) or alkylated (alkylhalides and silver oxide) according to standard processes. For that purpose, the 3- and 7-hydroxy groups may be protected temporarily in the form of a formate (removal with NH₃/MeOH) or of a p-methoxybenzyl ether (removal with DDQ).

Compounds according to general formula 2 are obtainable from epothilon A and B and also from their 3-O- and/or 7-O-protected derivatives by reduction, for example with NaBH₄ in methanol. If 3-OH and/or 7-OH are protected reversibly, then after acylation or alkylation and removal of the protecting groups there may be obtained 5-O-monosubstituted or 3,5- or 5,7-O-disubstituted derivatives of general formula 2.

Reactions of epothilon A and B with bifunctional electrophilic reagents, such as (thio)phosgene, (thio)carbonyldiimidazole, thionyl chloride or dialkylsilyl dichlorides or bistriflates yield compounds of general formula 3. Pyridine, trialkylamines, optionally together with DMAP or 2,6-lutidine in an aprotic solvent serve as auxiliary bases in the process. The 3,7-acetals of general formula 3 are produced by transacetalisation, for example of dimethylacetals in the presence of an acid catalyst.

Compounds according to general formula 4 are obtained from epothilon A and B or from 3-O- and/or 7-O—protected derivatives thereof by ozonolysis and reductive treatment, for example with dimethyl sulphide. The C-16-ketones may then be converted Into oximes, hydrazones or semicarbazones in accordance with standard processes known to the person skilled in the art. They are, moreover, converted into C-16-/C-17-olefins by Wittig, Wittig-Horner, Julia or Petersen olefination.

The 16-hydroxy derivatives according to general formula 5 are obtainable by reduction of the C-16-keto group, for example with an aluminium hydride or borohydride. If 3-OH and 7-OH are provided with suitable protecting groups, the 16-hydroxy derivatives may be either acylated or alkylated. The 3-OH and 7-OH groups are freed, for example, in the case of O-formyl by NH₃/MeOH and, in the case of O-p-methoxybenzyl, by DDQ.

The compounds of general formula 6 are obtained from derivatives of epothilon A and B, in which the 7-OH group has been protected by acyl or ether groups, by, for example, formylating, mesylating or tosylating the 3-OH group and then eliminating it by treatment with a base, for example DBU. The 7-OH group can be freed as described above.

Compounds of general formula 7 are obtained from epothilon A and B or from 3-OH- and 7-OH-protected derivatives thereof by basic hydrolysis, for example with NaOH in MeOH or MeOH/water. Preferably compounds of general formula 7 are obtained from epothilon A or B or from 3-OH— or 7-OH-protected derivatives thereof by enzymatic hydrolysis, especially with esterases or lipases. The carboxy group can be converted to an ester with a diazoalkane after protection of the 19-OH group by alkylation.

Moreover, compounds of formula 7 may be converted into compounds of formula 1 by lactonisation in accordance with the methods of Yamaguchi (trichlorobenzoyl chloride/DMAP), Corey (aldrithiol/triphenylphosphine) or Kellogg (omega-bromic acid/caesium carbonate). Relevant working methods may be found in Inanaga et al. in Bull. Chem. Soc. Japan, 52 (1979) 1989; Corey & Nicolaou in J. Am. Chem. Soc., 96 (1974) 5614; and Kruizinga & Kellogg in J. Am. Chem. Soc., 103 (1981) 5183.

To prepare the compounds according to the invention, it is also possible to start from epothilon C or D, where, for the derivatisation, reference may be made to the derivatisation methods described above. The 12,13-double bond may be selectively hydrogenated, for example catalytically or with diimine; or epoxidised, for example with dimethyldioxirane or with a peracid; or converted Into a dihalide, dipseudohalide or diazide.

The invention relates also to compositions for plant protection in agriculture, forestry and/or horticulture, consisting of one or more of the above-mentioned epothilon derivatives or consisting of one or more of the above-mentioned epothilon derivatives together with one or more common carrier(s) and/or diluent(s).

Finally, the invention relates to therapeutic compositions consisting of one or more of the above-mentioned compounds or of one or more of the above-mentioned compounds together with one or more common carrier(s) and/or diluent(s). These compositions may especially demonstrate cytotoxic activities and/or cause immunosuppression and/or be used to combat malignant tumours; they are particularity preferred as cytostatic agents.

The invention is illustrated and described hereinafter in greater detail by the description of a number of selected embodiments.

EXAMPLES Example 1

Compound 1a

20 mg (0.041 mmol) of epothilon A are dissolved in 1 ml of acetone, 50 μl (0.649 mmol) of trifluoroacetic acid are added and the reaction mixture is stirred overnight at 50° C. The reaction mixture is worked up by adding 1M phosphate buffer pH 7 and extracting the aqueous phase four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The raw product is purified by preparative layer chromatography (eluant:dichloromethane/acetone, 85:15).

Yield:

4 mg (19%) of isomer I

4 mg (19%) of isomer II

Isomer I

R₁ (dichloromethane/acetone, 85:15): 0.46 $\begin{matrix} \begin{matrix} {\quad {{{{IR}\quad ({film})\text{:}\quad {ny}} = {3440\quad \left( {m,b,{sh}} \right)}},{2946\quad \left( {s,{sh}} \right)},{1734\quad ({vs})},{1686\quad (m)},}} \\ {{{1456\quad (m)},{1375\quad (w)},{1256\quad \left( {s,{sh}} \right)},{1190\quad \left( {w,b,{sh}} \right)},}} \\ {{{1071\quad \left( {m,{sh}} \right)},{884\quad (w)},{735\quad (w)\quad {{cm}^{- 1}.}}}} \end{matrix} \\ \begin{matrix} {{{{MS}\quad \left( {{20/70}\quad {eV}} \right)\text{:}\quad {m/e}\quad (\%)} = {493\quad \left( {43\quad\left\lbrack {{M—H}_{2}O} \right\rbrack}^{+} \right)}},{394\quad (47)},{306\quad (32)},} \\ {{{206\quad (30)},{181\quad (40)},{166\quad (72)},{139\quad (100)},}} \\ {{{113\quad (19)},{71\quad (19)},{57\quad (24)},{43\quad {(24).}}}} \end{matrix} \end{matrix}$

Microanalysis: C₂₆H₃₉O₆NS calc.: 493.2498 for [M−H₂O]⁺

found: 493.2478

Isomer II

R₁(dichloromethane/acetone, 85:15): 0.22 IR  (film):  ny = 3484  (s, b, sh), 2942  (vs, sh), 1727  (vs), 1570  (w),   1456  (m), 1380  (m), 1265  (s), 1190  (w), 1069  (m),   975  (w)  cm⁻¹.MS  (20/70  eV):  m/e  (%) = 493  (21  [M—H₂O]⁺), 394  (12),   306  (46), 206  (37), 181  (63), 166  (99),   139  (100), 113  (21), 71  (23), 57  (33),   43  (28).

Microanalysis: C₂₆H₃₉O₆NS calc.: 493.2498 for [M−H₂O]⁺

found: 493.2475

Example 2

Compound 1b

55 mg (0.111 mmol) of epothilon A are dissolved in 0.5 ml of tetrahydrofuran, 0.5 ml of 1N hydrochloric acid is added, and the reaction mixture is stirred at room temperature for 30 minutes. 1N Phosphate buffer pH 7 is then added and the aqueous phase is extracted four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The raw product is purified by preparative layer chromatography (eluant: dichloromethane/methanol, 90:10). Yield: 19 mg (32%).

R₁ (dichloromethane/methanol, 90:10): 0.46

IR film): ny=3441 (s, br, sh), 2948 (s, sh), 1725 (vs, sh), 1462 (m),

1381 (w), 1265 (m), 1154 (w), 972 (m, br, sh) cm⁻¹.

UV (methanol): lambda_(max) (lg epsilon)=210 (4.29), 248 (4.11) nm.

MS (20/70 eV): m/e (%) 529 (13 [M⁺]), 494 (10), 342 (38), 306 (23),

194 (32), 164 (100), 140 (31), 113 (15), 57 (16).

Microanalysis: C₂₆H₄₀O₆CINS calc.: 529.2265 for [M³⁰],

found: 529.2280

Example 3

Compound 1c

25 mg (0.047 mmol) of 12-chloro-13-hydroxy-epothilon A (1b) are dissolved in 1 ml of dichloromethane, and 29 mg (0.235 mmol) of dimethylaminopyridine, 151 μl (1.081 mmol) of triethylamine and 20 μl (0.517 mmol) of 98% formic acid are added. The reaction mixture is cooled with ice/salt. When −15° C. has been reached, 40 μl (0.423 mmol) of acetic anhydride are added to the reaction mixture, which is stirred for 70 minutes at −15° C. Since thin-layer chromatography shows that the reaction is not complete, a further 6 mg (0.047 mmol) of dimethylaminopyridine, 7 μl (0.047 mmol) of triethylamine, 2 μl of 98% formic acid (0.047 mmol) and 4 μl (0.047 mmol) of acetic anhydride are added to the reaction mixture, which is stirred for 60 minutes. The reaction mixture is worked up by heating to room temperature, adding 1M phosphate buffer pH 7 and extracting the aqueous phase four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The raw product is purified by preparative layer chromatography (eluant: dichloromethane/acetone, 90:10).

Yield: 5 mg (1 8%).

R₁ (dichloromethane/acetone, 90:10): 0.67 IR  (film):  ny = 3497  (w, b, sh), 2940  (s, b, sh), 1725  (vs), 1468   (m, b, sh), 1379  (m), 1265  (s), 1253  (s), 1175  (vs), 972   (m, b, sh), 737  (s)  cm⁻¹.MS  (20/70  eV):  m/e  (%) = 613  (9  [M⁺]), 567  (43), 472  (63),   382  (23), 352  (21), 164  (100), 151  (33),   96  (31), 69  (17), 44  (26).

Microanalysis: C₂₉H₄₀O₉NSCl calc.: 613.2112 for [M⁺]

found: 613.2131

Example 4

Compound 1d

10 mg (0.020 mmol) of epothilon B are dissolved in 0.5 ml of tetrahydrofuran, 0.5 ml of 1N hydrochloric acid is added and the reaction mixture is stirred at room temperature for 30 minutes. 1M Phosphate buffer pH 7 is then added and the aqueous phase is extracted four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The raw product is purified by preparative layer chromatography (eluant:dichloromethane/acetone, 85:15).

Yield: 1 mg (9%).

R₁ (dichloromethane/acetone, 85:15): 0.38 $\begin{matrix} {{{{{MS}\quad \left( {{20/70}\quad {eV}} \right)}:\quad {{m/e}\quad (\%)}} = {543\quad \left( {3\quad\left\lbrack M^{+} \right\rbrack} \right)}},{507\quad (14)},{320\quad (19)},{234\quad (9)},} \\ {{{194\quad (17)},{182\quad (23)},{164\quad (100)},{140\quad (22)},}} \\ {{{113\quad (14)},{71\quad {(13).}}}} \end{matrix}$

Microanalysis: C₂₇H₄₂O₆NSCl calc.: 543.2421 for [M⁺]

found: 543.2405

Example 5

Compound 2a

100 mg (0.203 mmol) of epothilon A are dissolved in 4 ml of tetrahydrofuran/-1M phosphate buffer pH 7 (1:1), and sodium borohydride (150 mg=3.965 mmol) is added until the starting material has reacted completely according to thin-layer chromatography. Dilution with 1M phosphate buffer pH 7 is then carried out and the aqueous phase is extracted four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The raw product is purified by silica chromatography (eluant:dichloromethane/acetone, 95:5—gradient—to dichloromethane/acetone, 85:15).

Yield: (20%)

R₁ (dichloromethane/acetone, 75:25): 0.27 IR  (film):  ny = 3413  (s, b, sh), 2965  (vs, sh), 1734  (vs), 1458  (m, b, sh),   1383  (m, sh), 1264  (s, b, sh), 1184  (m, b, sh), 1059   (s, sh), 966  (s), 885  (w), 737  (m)  cm⁻¹.MS  (20/70  eV):  m/e  (%) = 495  (6  [M⁺]), 477  (8), 452  (12), 394  (9),   364  (16), 306  (49), 194  (19), 178  (35),   164  (100), 140  (40), 83  (21), 55  (27).

Microanalysis: C₂₆H₄₁O₆NS calc.: 495.2655 for [M⁺]

found: 495.2623

Example 6

Compound 3a-d (a-d are Stereoisomers)

100 mg (0.203 mmol) of epothilon A are dissolved in 3 ml of pyridine, 50 μl (0.686 mmol) of thionyl chloride are added and the reaction mixture is stirred at room temperature for 15 minutes. 1M Phosphate buffer pH 7 is then added and the aqueous phase is extracted four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The crude product is purified and the four stereoisomers 3a-d are separated by preparative layer chromatography (eluant: toluene/methanol, 90:10).

Compound 3a

Yield: 4 mg (12%)

R₁ (toluene/methanol, 90:10): 0.50 ${{{IR}\quad ({film})\text{:}\quad {ny}} = {2961\quad \left( {m,b,{sh}} \right)}},{1742\quad ({vs})},{1701\quad ({vs})},{1465\quad \left( {m,{sh}} \right)},\quad {1389\quad \left( {m,{sh}} \right)},{1238\quad \left( {s,{sh}} \right)},{1210\quad \left( {{vs},{sh}} \right)},{1011\quad \left( {s,{sh}} \right)},\quad {957\quad \left( {s,b,{sh}} \right)},{808\quad \left( {m,{sh}} \right)},{{768\quad \left( {s,{sh}} \right)\quad {{cm}^{- 1}.{UV}}\quad ({methanol})\text{:}\quad {{lambda}_{\max}\left( {\lg \quad {epsilon}} \right)}} = {210\quad (4.50)}},{248\quad (4.35)\quad {{nm}.\begin{matrix} {{{{MS}\quad \left( {{20/70}\quad {eV}} \right)\text{:}\quad {m/e}\quad (\%)} = {539\left( {40\left\lbrack M^{+} \right\rbrack} \right)}},{457(22)},{362(16)},{316(27)},} \\ {{{222(30)},{178(30)},{164(100)},{151(43)},}} \\ {{{96(38)},{69(29)},{55(28)},{43{(20).}}}} \end{matrix}}}$

Microanalysis: C₂₆H₃₇O₇NS₂ calc.: 539.2011 for [M⁺]

Compound 3b

Yield: 14 mg (13%)

R₁ (toluene/methanol, 90:10): 0.44 $\begin{matrix} {{{{IR}\quad ({film})\quad {ny}} = {2963\quad \left( {s,{br},{sh}} \right)}},{1740\quad ({vs})},{1703\quad (s)},{1510\quad (w)},1464} \\ {{\left( {m,{br},{sh}} \right),{1389\quad \left( {m,{sh}} \right)},{1240\quad \left( {s,{br},{sh}} \right)},{1142\quad (m)},1076}} \\ {{(w),{1037\quad (w)},{1003\quad (m)},{945\quad \left( {s,{br},{sh}} \right)},{806\quad \left( {m,{sh}} \right)},{775\quad (s)},}} \\ {{737\quad (m)\quad {{cm}^{- 1}.}}} \end{matrix}$

UV (methanol): lambda_(max)(lg epsilon)=211 (4.16), 250 (4.08) nm. $\begin{matrix} {{{{MS}\quad \left( {{20/70}\quad {eV}} \right)\text{:}\quad {m/e}\quad (\%)} = {539\quad \left( {27\quad\left\lbrack M^{+} \right\rbrack} \right)}},{475\quad (17)},{322\quad (41)},{306\quad (67)},} \\ {{{222\quad (16)},{206\quad (17)},{194\quad (19)},{178\quad (32)},}} \\ {{{164\quad (100)},{151\quad (33)},{125\quad (18)},{113\quad (15)},}} \\ {{{96\quad (39)},{81\quad (23)},{64\quad (58)},{57\quad (42)},{41\quad {(19).}}}} \end{matrix}$

Microanalysis: C₂₆H₃₇O₇NS₂ calc.: 539.2011 for [M⁺]

found: 539.1998

Compound 3c

Yield: 4 mg (4%)

R₁ (toluene/methanol, 90:10): 0.38 $\begin{matrix} {{{{MS}\quad \left( {{20/70}\quad {eV}} \right)\text{:}\quad {m/e}\quad (\%)} = {539\quad \left( {69\quad\left\lbrack M^{+} \right\rbrack} \right)}},{322\quad (22)},{306\quad (53)},{222\quad (36)},} \\ {{{178\quad (31)},{164\quad (100)},{151\quad (41)},{96\quad (25)},}} \\ {{{81\quad (20)},{69\quad (26)},{55\quad (25)},{41\quad (25)}}} \end{matrix}$

Microanalysis: C₂₆H₃₇O₇NS₂ calc.: 539.2011 for [M⁺]

found: 539.2001

Compound 3d

Yield: 1 mg (1%)

R₁ (toluene/methanol, 90:10): 0.33 $\begin{matrix} {{{{{MS}\left( {{20/70}\quad {eV}} \right)}\text{:}\quad {m/{e(\%)}}} = {539\left( {69\left\lbrack M^{+} \right\rbrack} \right)}},{322(35)},{306(51)},} \\ {{{222(41)},{178(31)},{164(100)},{151(46)},}} \\ {{{96(31)},{81(26)},{69(34)},{55(33)},{41(35)}}} \end{matrix}$

Microanalysis: C₂₆H₃₇O₇NS₂ calc.: 539.2011 for [M⁺]

found: 539.1997

Example 7

Compound 4a

10 mg (0.020 mmol) of epothilon A are dissolved in 2 ml of dichloromethane, cooled to −70° C. and then treated with ozone for 5 minutes until there is a slight blue coloration. 0.5 ml of dimethyl sulphide is subsequently added to the resulting reaction mixture, which is heated to room temperature. The reaction mixture is worked up by freeing it of solvent and finally by preparative layer chromatography (eluant:dichloromethane/-acetone/methanol, 85:10:5).

Yield: 5 mg (64%)

R₁ (dichloromethane/acetone/methanol, 85:10:5): 0.61 $\begin{matrix} {{{{{IR}\quad ({film})}:\quad {ny}} = {3468\quad \left( {s,{br},{sh}} \right)}},{2947\quad \left( {s,{br},{sh}} \right)},{1734\quad \left( {{vs},{sh}} \right)},{1458\quad (w)},} \\ {{{1380\quad (w)},{1267\quad (w)},{1157\quad (w)},{1080\quad (w)},{982\quad (w)\quad {{cm}^{- 1}.}}}} \end{matrix}$

UV (methanol): lambda_(max) (lg epsilon)=202 (3.53) nm. $\begin{matrix} {{{{{MS}\quad \left( {{20/70}\quad {eV}} \right)}:\quad {{m/e}\quad (\%)}} = {398\quad \left( {2\quad\left\lbrack M^{+} \right\rbrack} \right)}},{380\quad (4)},{267\quad (14)},{249\quad (17)},} \\ {{{211\quad (20)},{193\quad (26)},{171\quad (34)},{139\quad (34)},}} \\ {{{111\quad (40)},{96\quad (100)},{71\quad (48)},{43\quad {(50).}}}} \end{matrix}$

Microanalysis: C₂₁H₃₄O₇: calc.: 398.2305 for [M⁺]

found: 398.2295

Example 8

Compound 6a

10 mg (0.018 mmol) of 3,7-di-O-formyl-epothilon A are dissolved in 1 ml of dichloromethane, 27 μl (0.180 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are added and the reaction mixture is stirred at room temperature for 60 minutes. The reaction mixture is worked up by adding 1M sodium dihydrogen phosphate buffer pH 4.5 and extracting the aqueous phase four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. After the solvent has been removed, the resulting raw product is dissolved in 1 ml of methanol, 200 μl of an ammoniacal methanol solution (2 mmol NH₃/ml methanol) are added and the mixture is stirred overnight at room temperature. For separation, the solvent is removed in vacuo.

Yield: 4 mg (22%)

R₁ (dichloromethane/acetone, 85:15): 0.46 $\begin{matrix} {{{{{IR}\quad ({film})}:\quad {ny}} = {3445\quad \left( {w,{br},{sh}} \right)}},{2950\quad \left( {{vs},{br},{sh}} \right)},{1717\quad \left( {{vs},{sh}} \right)},{1644\quad (w)},} \\ {{{1466\quad \left( {m,{sh}} \right)},{1370\quad \left( {m,{sh}} \right)},{1267\quad \left( {s,{br},{sh}} \right)},{1179\quad \left( {s,{sh}} \right)},}} \\ {{{984\quad \left( {s,{sh}} \right)},{860\quad (w)},{733\quad (m)\quad {{cm}^{- 1}.}}}} \end{matrix}$

UV (methanol): lambda_(max) (lg epsilon)=210 (4.16) nm. $\begin{matrix} {{{{{MS}\quad \left( {{20/70}\quad {eV}} \right)}:\quad {{m/e}\quad (\%)}} = {475\quad \left( {28\quad\left\lbrack M^{+} \right\rbrack} \right)}},{380\quad (21)},{322\quad (37)},{318\quad (40)},} \\ {{{304\quad (66)},{178\quad (31)},{166\quad (100)},{151\quad (29)},}} \\ {{{140\quad (19)},{96\quad (38)},{81\quad (20)},{57\quad {(26).}}}} \end{matrix}$

Microanalysis: C₂₆H₃₇O₅NS calc.: 475.2392 for [M⁺]

found: 475.2384

Example 9

Compound 6b

50 mg (0.091 mmol) of 3,7-di-O-formyl-epothilon A are dissolved in 1 ml of dichloroethane, 2 ml (0.013 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) are added and the reaction mixture is stirred for 12 hours at 90° C.

The reaction mixture is worked up by adding 1M sodium dihydrogen phosphate buffer pH 4.5 and extracting the aqueous phase four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The raw product, consisting of two compounds, Is purified by preparative layer chromatography (eluant:dichloro-methane/acetone, 90:10).

Yield: 7 mg (15%)

Substance Code

R₁ (dichloromethane/acetone, 90:10): 0.62 $\begin{matrix} {{{{{IR}\quad ({film})}:\quad {ny}} = {2951\quad \left( {m,{br},{sh}} \right)}},{1723\quad ({vs})},{1644\quad \left( {w,{br},{sh}} \right)},{1468\quad (w)},} \\ {{{1377\quad (w)},{1271\quad \left( {m,{br},{sh}} \right)},{1179\quad (s)},{987\quad \left( {m,{br},{sh}} \right)},}} \\ {{735\quad \left( {w,{br},{sh}} \right)\quad {{cm}^{- 1}.}}} \end{matrix}$

UV (methanol): lambda_(max) (lg epsilon)=210 (4.44) nm. $\begin{matrix} {{{{{MS}\quad \left( {{20/70}\quad {eV}} \right)}:\quad {{m/e}\quad (\%)}} = {503\quad \left( {68\quad\left\lbrack M^{+} \right\rbrack} \right)}},{408\quad (58)},{390\quad (32)},{334\quad (25)},} \\ {{{316\quad (34)},{220\quad (21)},{206\quad (27)},{194\quad (20)},}} \\ {{{181\quad (33)},{164\quad (100)},{151\quad (34)},{139\quad (28)},}} \\ {{{113\quad (20)},{96\quad (82)},{81\quad (33)},{67\quad (24)},{55\quad (26)},}} \\ {{43\quad {(22).}}} \end{matrix}$

Microanalysis: C₂₇H₃₇O₆NS calc.: 503.2342 for [M⁺]

found: 503.2303

Example 10

Compound 6c

5 mg (0.009 mmol) of 3,7-di-O-acetyl-epothilon A are dissolved in 1 ml of methanol, 150 μl of an ammoniacal methanol solution (2 mmol NH₃/ml methanol) are added and the reaction mixture is stirred overnight at 50° C. For separation, the solvent is removed in vacuo. The raw product is purified by preparative layer chromatography (eluant: toluene/methanol, 90:10).

Yield: 3 mg (67%)

R₁ (dichloromethane/acetone, 90:10): 0.55 $\begin{matrix} {{{{{IR}\quad ({film})}:\quad {ny}} = {2934\quad \left( {s,b,{sh}} \right)}},{1719\quad \left( {{vs},b,{sh}} \right)},{1641\quad (m)},{1460\quad \left( {m,{sh}} \right)},} \\ {{{1372\quad \left( {s,{sh}} \right)},{1237\quad \left( {{vs},b,{sh}} \right)},{1179\quad \left( {s,{sh}} \right)},{1020\quad (s)},}} \\ {{{963\quad \left( {s,{sh}} \right)},{737\quad ({vs})\quad {{cm}^{- 1}.}}}} \end{matrix}$

UV (methanol): lambda_(max) (lg epsilon)=210 (4.33) nm. $\begin{matrix} {{{{{MS}\quad \left( {{20/70}\quad {eV}} \right)}:\quad {{m/e}\quad (\%)}} = {517\quad \left( {57\quad\left\lbrack M^{+} \right\rbrack} \right)}},{422\quad (58)},{318\quad (31)},{194\quad (20)},} \\ {{{181\quad (34)},{166\quad (100)},{151\quad (31)},{96\quad (96)},}} \\ {{{81\quad (32)},{69\quad (27)},{55\quad (29)},{43\quad {(69).}}}} \end{matrix}$

Microanalysis: C₂₈H₃₉O₆NS calc.: 517.2498 for [M⁺]

found: 517.2492

Example 11

Compound 7a

20 mg (0.041 mmol) of epothilon A are dissolved in 0.5 ml of methanol, 0.5 ml of 1 N sodium hydroxide solution is added and the reaction mixture is stirred at room temperature for 5 minutes.

The reaction mixture is worked up by adding 1M phosphate buffer pH 7 and extracting the aqueous phase four times with ethyl acetate. The combined organic phases are washed with saturated sodium chloride solution, dried over sodium sulphate and freed of solvent. The raw product is purified by preparative layer chromatography (eluant: dichloromethane/methanol, 85:15).

Yield :11 mg (52%)

R₁ (dichloromethane/methanol, 85:15): 0.92 $\begin{matrix} {{{{{IR}\quad ({film})}:\quad {ny}} = {3438\quad \left( {s,{br},{sh}} \right)}},{2971\quad \left( {{vs},{br},{sh}} \right)},{1703\quad ({vs})},{1507\quad (m)},} \\ {{{1460\quad \left( {s,{sh}} \right)},{1383\quad \left( {m,{sh}} \right)},{1254\quad (w)},{1190\quad \left( {w,{br},{sh}} \right)},}} \\ {{{1011\quad \left( {w,{br},{sh}} \right)},{866\quad \left( {w,{br}} \right)},{729\quad (s)\quad {{cm}^{- 1}.}}}} \end{matrix}$

MS (20/70 eV): m/e (%)=423 (0.1 [M⁺]), 323 (4), 168 (89), 140 (100), 85 (31), 57 (67).

Microanalysis: C₂₃H₃₇O₄NS calc.: 423.2443 for [M⁺]

found: 423.2410

Example 12

Compound 7b

5 mg (0.009 mmol) of 7-O-acetyl-epothilon A are dissolved in 1 ml of methanol, 200 μl of an ammoniacal methanol solution (2 mmol NH₃/ml methanol) are added and the reaction mixture is stirred at 50° C. for two days. For separation, the solvent is removed in vacuo. The raw product is purified by preparative layer chromatography (eluant: toluene/methanol, 90:10).

Yield: 3 mg (59%)

R₁ (dichloromethane/methanol, 90:10): 0.63 $\begin{matrix} {{{{{IR}\quad ({film})}:\quad {ny}} = {3441\quad \left( {m,b,{sh}} \right)}},{2946\quad \left( {s,{sh}} \right)},{1732\quad ({vs})},{1600\quad (w)},} \\ {{{1451\quad (m)},{1375\quad (m)},{1246\quad \left( {s,b,{sh}} \right)},{1013\quad \left( {m,b,{sh}} \right)\quad {{cm}^{- 1}.}}}} \end{matrix}$

UV (methanol): lambda_(max) (lg epsilon)=211 (3.75), 247 (3.59) nm. ${{{MS}\left( {{20/70}\quad {eV}} \right)}\text{:}\quad {m/e}\quad (\%)} = \begin{matrix} \begin{matrix} \quad \\ {{567\left( {1\left\lbrack M^{+} \right\rbrack} \right)},{465(4)},{422(7)},{388(5)},{194(5)},{182(7)},} \end{matrix} \\ {{168(65)},{164(17)},{140(100)},{97(10)},{71(22)},{43{(27).}}} \end{matrix}$

Microanalysis: C₂₉H₄₅O₈NS calc.: 567.2866 for [M⁺]

found: 567.2849

Example 13

50 mg of epothilon A are dissolved in 20 μl of dimethyl sulphoxide and diluted with 30 ml of phosphate buffer (pH 7.1, 30 mM). After the addition of 5 mg of pig liver esterase (Boehringer Mannheim), the mixture is stirred for 2 days at 30° C. The mixture is acidified to pH 5 with 2N HCl and the epothilonic acid 7 is extracted with ethyl acetate. The organic phase is dried with sodium sulphate and concentrated to dryness by evaporation in vacuo. Yield 48 mg (96%).

Example 14

48 mg of epothilonic A acid 7 are dissolved in 6 ml of abs. THF and, with stirring, 40 p1 μf triethylamine and 16 μl of 2,4,6-trichlorobenzoyl chloride are added. After 15 minutes, the precipitate is removed by filtration and the filtrate is added dropwise, within a period of 15 minutes, with rapid stirring, to a boiling solution of 20 mg of 4-dimethylaminopyridine in 200 ml of abs. toluene. After a further 10 minutes, the mixture is concentrated by evaporation in vacua and the residue is partitioned between ethyl acetate/citrate buffer (pH 4). After separation by preparative HPLC, the evaporation residue of the organic phase yields 15 mg of epothilon A.

Example 15

Epothilons C and D as Starting Materials

A. Production Strain and Culture Conditions Corresponding to the Epothilon Basic Patent.

B. Production using DSM 6773

75 litres of culture are grown as described in the basis patent and are used for inoculation in a production fermenter containing 700 litres of production medium consisting of 0.8% starch, 0.2% glucose, 0.2% soya flour, 0.2% yeast extract, 0.1% CaCl₂×2H₂O, 0.1#MgSO₄×7H₂O, 8 mg/litre of Fe-EDTA, pH=7.4 and optionally 15 litres of Amberlite XAD-16 adsorber resin. Fermentation takes 7 to 10 days at 30° C., with aeration with 2 m³ air/h. The pO₂ is maintained at 30% by regulating the rotary speed.

C. Isolation

The adsorber resin is separated from the culture using a 0.7 m₂ 100-mesh process filter and is freed of polar impurities by washing with 3 bed volumes of water/methanol 2:1. Elution with 4 bed volumes of methanol yields a raw extract which is concentrated by evaporation in vacuo until the aqueous phase occurs. That is then extracted three times with the same volume of ethyl acetate. Concentration of the organic phase by evaporation yields 240 g of raw extract which is partitioned between methanol and heptane in order to separate lipophilic impurities. From the methanolic phase there are obtained by evaporation in vacuo 180 g of isolate which is fractionated in three portions over Sephadex LH-20 (20×100 cm column, 20 ml/min methanol). The epothilons are contained in the fraction which is eluted in the retention time from 240 to 300 minutes and which comprises a total of 72 g. To separate the epothilons, chromatography is carried out in three portions on Lichrosorb RP-18 (15 μm, 10×40 cm column, eluant 180 ml/min methanol/water 65:35). After epothilon A and B there are eluted epothilon C at R₁=90-95 min and epothilon D at R₁=100-110 min, which are obtained, after evaporation in vacuo, in each case in a yield of 0.3 g of a colourless oil.

D. Physical Properties

Epothilon C R═H

Epothilon D R═CH₃

Epothilon C

C₂₆H₃₉NO₆S [477]

ESI-MS : (positive ions): 478.5 for [M+H]⁺

1H and 13C, see NMR table

TLC: R₁=0.82

TLC aluminium foil 60 F 254 Merck, eluant:dichloromethane/methanol=9:1

Detection: UV extinction at 254 nm. Spraying with vanillin/sulphuric acid reagent, blue-grey coloration on heating to 1 20° C.

HPLC: R₁=11.5 min

Column: Nucleosil 100 C-18 7 μm, 125×4 mm.

Eluant: methanol/water=65:35

Flow rate: 1 ml/min

Detection: diode array

Epothilon D

C₂₇H₄₁NO₅S[491]

ESI-MS: (positive ions): 492.5 for [M+H]⁺

1H and 13C, see NMR table

TLC: R₁=0.82

TLC aluminium foil 60 F 254 Merck, eluant:dichloromethane/methanol=9:1

Detection: UV extinction at 254 nm. Spraying with vanillin/sulphuric acid reagent, blue-grey coloration on heating to 120° C.

HPLC: R₁=15.3 min

Column: Nucleosil 100 C-18 7 μm, 125×4 mm

Eluant: methanol/water=65:35

Flow rate: 1 ml/min

Detection: diode array

TABLE ¹H and ¹³C NMR data of epothilon C and epothilon D in [D₆]DMSO at 300 MHz Epothilon C Epothilon D δ C δ δ C δ H atom (ppm) atom (ppm) (ppm) atom (ppm) 1 170.3 1 170.1  2-Ha 2.38 2 38.4 2.35 2 39.0  2-Hb 2.50 3 71.2 2.38 3 70.8  3-H 3.97 4 53.1 4.10 4 53.2  3-OH 5.12 5 217.1 5.08 5 217.4  6-H 3.07 6 45.4 3.11 6 44.4  7-H 3.49 7 75.9 3.48 7 75.5  7-OH 4.46 8 35.4 4.46 8 36.3  8-H 1.34 9 27.6 1.29 9 29.9  9-Ha 1.15 10 30.0 1.14 10 25.9  9-Hb 1.40 11 27.6 1.38 11 31.8* 10-Ha 1.15* 12 124.6 1.14* 12 138.3 10-Hb 1.35* 13 133.1 1.35* 13 120.3 11-Ha 1.90 14 31.1 1.75 14 31.6* 11-Hb 2.18 15 76.3 2.10 15 76.6 12-H 5.38** 16 137.3 18 137.2 13-H 5.44** 17 119.1 5.08 17 119.2 14-Ha 2.35 18 152.1 2.30 18 152.1 14-Hb 2.70 19 117.7 2.65 19 117.7 15-H 5.27 20 164.2 5.29 20 164.3 17-H 6.50 21 16.8 6.51 21 18.9 19-H 7.35 22 20.8 7.35 22 19.7 21-H₃ 2.65 23 22.6 2.65 23 22.5 22-H₃ 0.94 24 16.7 0.90 24 16.4 23-H₃ 1.21 25 18.4 1.19 25 18.4 24-H₃ 1.06 27 14.2 1.07 26 22.9 25-H₃ 0.90 0.91 27 14.1 26-H₃ 1.63 27-H₃ 2.10 2.11 *, **allocation interchangeable

Example 16

Epothilon A and 12,13-bisepi-epothilon A from Epothilon C

50 mg of epothilon C are dissolved in 1.5 ml of acetone, and 1.5 ml of a 0.07M solution of dimethyldioxirane in acetone are added. After 6 hours' standing at room temperature, concentration by evaporation in vacuo is carried out and separation is effected by preparative HPLC on silica gel (eluant: methyl tert-butyl ether/petroleum ether/methanol 33:66:1).

Yield:

25 mg of epothilon A, R₁=3.5 min (analyt. HPLC, 7 μm, 4×250 mm column,

eluant see above, flow rate 1.5 ml/min) and

20 mg of 12,13-bisepi-epothilon A, R₁=3.7 min, ESI-MS (pos. ions)

m/z=494 [M+H]⁺

¹H-NMR in [D₄] methanol, selected signals:

delta=4.32 (3-H), 3.79 (7-H), 3.06 (12-H),

3.16 (13-H), 5.54 (15-H), 6.69 (17-H), 1.20

(22-H), 1.45 (23-H).

12,13-bisepi-epothilon A R═H

Example 17

[1S-[1R*,3R*(E),10S*,11S*,12R*,16S*]]-11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadec-6-ene-5,9-dione

A. [1S-[1R*,3R*(E),7R*,10S*,11S*,12R*,16S*]]-7,11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione, 7,11-diformate.

Formic acid (0.95 ml, 25 mmol, 5.0 equiv), 4-N,N-dimethylaminopyridine (1.3 g, 11 mmol), and triethylamine (7.0 ml, 49 mmol) were added to a solution of epothilone B (2.5 g, 5.0 mmol) in CH₂Cl₂ (100 ml). The reaction mixture was cooled to −15° C. and acetic anhydride (2.3 ml, 25 mmol) was added over 5 minutes. The reaction mixture was stirred for 15 minutes at −15° C. then warmed to room temperature and stirred for 30 minutes. The reaction mixture was quenched with pH 7.0 phosphate buffer, and the organic layer extracted with CH₂Cl₂ (3×100 ml). The combined organic extracts were washed with 1 N HCl (1×100 ml) and 10% NaHCO₃ (1×100 ml). The organic layer was dried (Na₂SO₄), filtered, and concentrated under vacuum to give Compound A (2.8 g, 100%), a glassy solid, which was used without further purification.

MS (M+H)⁺564

TLC: Rf=0.71 (9/1 CH₂Cl₂/acetone, visualization with UV)

B. [1S-[1R*,3R*(E),10S*,11S*,12R*,16S*]]-11-Formyloxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadec-6-ene-5,9-dione, 11-formate.

1,8-Diazabicyclo[5.4.0]undec-7-ene (7.3 ml, 49 mmol, 10 equiv), was added over 5 minutes to a solution of Compound A (2.8 g, 4.9 mmol) in CH₂Cl₂ (140 ml) at room temperature. The reaction mixture was stirred at room temperature for one hour. The reaction mixture was quenched with pH 4.0 phosphate buffer and extracted with CH₂Cl₂ (3×200 ml). The combined organic extracts were washed with 10% NaHCO₃ (1×200 ml). The organic layer was dried (Na₂SO₄), filtered, and concentrated under vacuum to afford Compound B (2.5 g, 100%), a glassy solid, which was used without further purification.

MS (M+H)⁺518

TLC: Rf=0.76 (9/1 CH₂Cl₂/acetone, visualization with UV)

C. [1S-[1R*,3R*(E),10S*,11S*,12R*,16S*]]-11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadec-6-ene-5,9-dione.

2M Ammonia in methanol (20 ml, 40 mmol) was added to a solution of Compound B (2,5 g, 4.8 mmol) in methanol (100 ml) at room temperature. The reaction mixture was stirred at room temperature for four hours. The reaction mixture was concentrated under vacuum. The residue was purified by flash chromatography (eluting with 19/1 CH₂Cl₂/acetone) to afford the title compound (2.1 g, 89%), as a glassy white solid.

MS (M+H)⁺490

TLC: Rf=0.41 (9/1 CH₂Cl₂/acetone, visualization with UV)

Elemental analysis for C₂₇H₃₉NO₈S.0.22H₂O Calc: C, 65.70; H, 8.05; N, 2.84 Found: C, 65.69; H, 8.12; N, 2.77. 

What is claimed is:
 1. A compound of the formula:

wherein R is hydrogen or C₁₋₄alkyl; X is oxygen, NOR³, N—NR⁴R⁵, or N—NHCONR⁴R⁵; each of R¹, R², R³, R⁴, and R⁵, independently from the others, is hydrogen, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl substituted by C₁₋₆alkoxy, C₁₋₆alkyl, hydroxy, or halogen, or R⁴ and R⁵ together are C₂₋₆alkylene group, and the alkyl, alkylene, and acyl groups contained in the radicals are straight-chain or branched radicals; and each of Y and Z, independently from the other, is hydrogen, halogen, pseudo-halogen, OH, O—(C₁₋₆)alkyl, O—(C₁₋₆)acyl or O-benzoyl, or Y and Z together are oxygen or a carbon-carbon bond.
 2. The compound according to claim 1 in which each of R, R¹, and R² is hydrogen, X is oxygen, and Y and Z together are oxygen.
 3. A compound of the formula:

wherein R is hydrogen or C₁₋₄alkyl; X is hydrogen, C₁₋₈alkyl, C₁₋₈acyl, benzyl, benzoyl, or cinnamoyl; each of R¹ and R², independently from the other, is hydrogen, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl substituted by C₁₋₆alkoxy, C₁₋₆alkyl, hydroxy, or halogen, and the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals; and each of Y and Z, independently from the other, is hydrogen, halogen, pseudo-halogen, OH, O—(C₁₋₆)alkyl, O—(C₁₋₆)acyl or O-benzoyl, or Y and Z together are oxygen or a carbon-carbon bond.
 4. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to either claim 1 or claim 3 in combination with a pharmaceutically acceptable carrier.
 5. A pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula:

wherein R is hydrogen or C₁₋₄alkyl; each of R¹, R², and R³, independently from the others, is hydrogen, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl substituted by C₁₋₆alkoxy, C₁₋₆alkyl, hydroxy, or halogen, and the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals; and each of Y and Z, independently from the other, is hydrogen, halogen, pseudo-halogen, OH, O—(C₁₋₆)alkyl, O—(C₁₋₆)acyl or O-benzoyl, or Y and Z together are oxygen or a carbon-carbon bond;

wherein R is hydrogen or C₁₋₄alkyl; X is —C(O)—, —C(S)—, —S(O)—, —CR¹R²—, or —SiR²— in which each of R¹ and R², independently from the other, is hydrogen, C₁₋₆alkyl, C₁₄acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl substituted by C₁₋₆alkoxy, C₁₋₆alkyl, hydroxy, or halogen, or R¹ and R² taken together are C₂₋₆alkylene, and the alkyl, alkylene, and acyl groups contained in the radicals are straight-chain or branched radicals; and each of Y and Z, independently from the other, is hydrogen, halogen, pseudo-halogen, OH, O—(C₁₋₆)alkyl, O—(C₁₋₆)acyl or O-benzoyl, or Y and Z together are oxygen or a carbon-carbon bond;

wherein R is hydrogen or C₁₋₄alkyl; R¹ is hydrogen, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl substituted by C₁₋₆alkoxy, C₁₋₆alkyl, hydroxy, or halogen, and the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals; and each of Y and Z, independently from the other, is hydrogen, halogen, pseudo-halogen, OH, O—(C₁₋₆)alkyl, O—(C₁₋₆)acyl or O-benzoyl, or Y and Z together are oxygen or a carbon-carbon bond; or

wherein R is hydrogen or C₁₋₄alkyl; each of R¹, R², R³, and R⁴, independently from the others, is hydrogen, C₁₋₆alkyl, C₁₋₆acyl, benzoyl, C₁₋₄trialkylsilyl, benzyl, phenyl, or benzyl or phenyl substituted by C₁₋₆alkoxy, C₁₋₆alkyl, hydroxy, or halogen, and the alkyl and acyl groups contained in the radicals are straight-chain or branched radicals, and each of Y and Z, independently from the other, is hydrogen, halogen, pseudo-halogen, OH, O—(C₁₋₆)alkyl, O—(C₁₋₆)acyl or O-benzoyl, or Y and Z together are oxygen or a carbon-carbon bond; in combination with a pharmaceutically acceptable carrier.
 6. The composition according to claim 4 in which in said compound, each of R, R¹, and R² is hydrogen, X is oxygen, and Y and Z together are oxygen. 